With the increasing interest in well-being or a healthy life, tagatose has been proposed as an alternative to sugar as it has less side effects and sugar is one of the major factors causing various adult diseases. Tagatose is the isomer of galactose and is known to have fructose-like physiochemical properties. Tagatose is a natural low-calorie sugar, and has recently been approved by the FDA in the USA as GRAS (Generally Recognized As Safe), so it is now allowed to be added as a sweetener to foods, beverages, health foods, diet additives, etc.
GRAS indicates a substance that is generally recognized as safe, which is judged by specialized people having enough experience and skills through scientific procedure and examination under the indicated conditions and purpose of use. GRAS is a unique system used only in the USA to evaluate the safety of foods and food chemical substances (under certain conditions) but it is recognized world-wide.
Tagatose is produced by either isomerization, which is a chemical method using a catalyst to produce an isomer, of galactose or a biological method using isomerase to convert galactose enzymatically.
One of the biological methods well-known to those in the art is to convert aldose or aldose derivatives into ketose or ketose derivatives by using an enzyme. The isomerization of galactose to tagatose by using arabinose isomerase is generally carried out thermodynamically at high temperature and exhibits a proportionally high conversion rate. Therefore, developing an enzyme that works stably at high temperature and a method of preparing tagatose using the same are key techniques for the industrial application thereof based on the biological conversion of tagatose using an isomerase. By screening thermophilic bacteria derived arabinose isomerases, an industrially applicable thermophilic isomerase has been tried, and efforts have also been made by many research teams to establish an isomerization process using the same.
In Korea, an enzymatic isomerization method using arabinose isomerase has been developed by Tong Yang Confectionery Corp. According to the method, the arabinose isomerase derived from E. coli was homogeneously expressed in E. coli by recombinant technology. This recombinant isomerase was reacted at 30° C. for 24 hours to convert galactose into tagatose, and at this time the conversion rate was 25%, indicating that both thermostability and conversion yield were very low (Korean Patent Application No. 99-16118). Professor Oh, Deok-keun and his colleagues (Sejong University) succeeded in heterogeneous expression of arabinose isomerase originating from Geobacillus stearothermophilus in E. coli and based on that they proposed an isomerization procedure at high temperature to convert galactose into tagatose. The present inventors succeeded in cloning a novel arabinose isomerase gene from Thermotoga neapolitana and producing it in E. coli, and further developed a method of manufacturing tagatose at hyper-thermophilic condition using the enzyme (Korean Patent No. 10-0443865).
The production of tagatose using a derived arabinose isomerase derived from thermophiles still depends on the method of using a recombinant enzyme mass-expressed in recombinant E. coli or the isomerization of galactose to tagatose using a host containing the recombinant enzyme. However, this biological production of tagatose using recombinant E. coli is not appropriate for the production of tagatose as a food ingredient. To produce tagatose as a food additive, arabinose isomerase expressed in a host which is a GRAS microorganism appropriate for the mass-production of the same is essential.
Corynebacterium is an industrial microorganism that has been used for the production of chemical compounds applicable to a variety of fields including L-lysine, L-threonine and various nucleic acid containing feeds, medical supplies and foods. The Corynebacterium is a GRAS (Generally Recognized As Safe) strain, which favors gene manipulation and mass production. In addition, this strain is highly stable under the various process conditions and has a comparatively stable cell membrane, compared with other bacteria. Thus, this strain remains stable even under high osmotic pressure generated by a high concentration of galactose.
The present inventors found that thermophilic arabinose isomerase originating from the hyperthermophile Thermotoga neapolitana is overexpressed as an active form in a GRAS Corynebacterium sp. strain, and further completed this invention by developing a novel art to induce tagatose isomerization from the high concentration of galactose by using the expressed recombinant enzymes from Corynebacterium species.